Rapid, instrument-free colorimetric quantification of DNA using Nile Blue

Abstract

The use of nucleic acid tests (NAT) for sensitive and rapid detection of pathogens relevant to human health has increased due to the ubiquity of nucleic acid amplification techniques such as polymerase chain reaction. The use of such tools for detection of amplified nucleic acid (NA) in field and clinical settings is limited by the need for complex instrumentation and trained users. To address these limitations we developed a rapid, robust, and instrument-free colorimetric detection method for nucleic acids using a visible region dye, Nile Blue (NB). NB is a cationic benzophenoxazine dye with well-known binding interactions with NA and has been used in instrumental methods for DNA quantification. When combined with dsDNA, the color of NB shifts from blue to purple. Images of this color shift are collected and are subjected to image analysis. Observed changes in the red and green colorimetric intensities are linked to the ratio of dsDNA to NB. By titrating solutions of dsDNA against a series of NB concentrations, we found it possible to quantitate dsDNA at concentrations ranging from 10–100 μg mL⁻¹ using a k-means cluster analysis method. This range is comparable to that of NA concentrations quantified using gold-standard UV-Visible spectroscopy and to the concentrations of NA in biological samples after amplification. Unknown concentrations of dsDNA from yeast extracts were correctly identified within ±5 μg mL⁻¹ of true concentration. Preliminary experiments demonstrate use of the developed NB method on paper-based analytical devices. As an instrument-free detection method, NB allows for rapid and robust quantification of dsDNA in field settings.